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Development and Characterization of a Novel Fusion Protein Composed

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Development and Characterization of a Novel Fusion Protein Composed J. Biochem. 133, 59-66 (2003) DOI : 10.1093/jb/mvg005 Development and Characterization of a Novel Fusion Protein Composed of a Human IgG1 Heavy Chain Constant Region and a Single-Chain Fragment Variable Antibody against Venezuelan Equine Encephalitis Virus Wei-Gang Hu1,2, Azhar Z. Alvi1, Damon Chau1 , Jeffrey E. Coles1, R. Elaine Fulton1, M avanur R. Suresh2 and Les P. Nagata*,1 1Chemical Biological Defence Section, Defence Research & Development Canada -Suffield, Box 4000, Station Main, Medicine Hat, Alberta, Canada TZA 8K6; and 'Faculty of Pharmacy and Pharmaceutical Sciences, University of Alb erta, Edmonton, Alberta, Canada T6G 2N8 Received August 28, 2002; accepted October 25 , 2002 Murine monoclonal antibody 1A4A1 has been shown to recognize a conserved neu tralizing epitope of envelope glycoprotein E2 of Venezuelan equine encephalitis virus. It is a potential candidate for development of a second generation antibody for both immunodiagnosis and immunotherapy . In order to minimize the immunogenic ity of murine antibodies and to confer human immune effector functions on murine antibodies, a recombinant gene fusion was constructed . It encoded a human IgGl heavy chain constant region and a single-chain fragment variable antibody of 1A4A1 . After expression in bacteria as inclusion bodies, the recombinant antibody was puri fied and refolded in vitro. The recombinant soluble antibody was demonstrated to retain high antigen-binding affinity to Venezuelan equine encephalitis virus and to possess some human IgG crystallizable fragment domain functions, such as recogni tion by protein G and human complement Clq binding. On non-reducing and reduc ing gel electrophoresis analysis of proteolytic fragments of the recombinant antbody , di sulfide bond formation was found in the hinge region of the antibody. From these data, it was concluded that the recombinant antibody was capable of antigen recogni tion, and retained several functional activities. This work forms the basis for charac terization of the recombinant antibody as to efficacy in vivo. Key words: antibody engineering, characterization, chimeric antibody, Venezuelan equine encephalitis virus. Abbreviations: Ab, antibody; ABTS, 2,2•Œ-azino-di-(3-ethyl-benzthiazoline-sulfonic acid) diammonium salt; ADCC , Ab -dependent cellular cytotoxicity; CH , heavy chain constant region; ELISA, enzyme-linked immunosorbent assay; Fab, antigen-binding fragment; (Fab•Œ)2, fragment composed of two Fab units and a hinge region; Fe , crystalli zable fragment; HRP, horseradish peroxidase; IMAC, immobilized metal affinity chromatography; IPTG , isopro pyl ƒÀ-D-thiogalactopyranoside; MAb, monoclonal antibody; NTA, nitrilotriacetic acid; PBS, phosphate-buffered saline; PBST, PBS containing 0.1% Tween-20; ScFv, single-chain fragment variable; VEE , Venezuelan equine encephalitis virus; VL, light chain variable region; VH, heavy chain variable region; WEE , Western equine encephalitis virus. Venezuelan equine encephalitis virus (VEE), a member of against VEE have been developed, these products are far the alphavirus genus of the family Togaviridae, is an from satisfactory. Approximately 20% of live-attenuated important pathogen for epidemics in humans, and epi TC-83 vaccine recipients fail to develop neutralizing anti zootics in rodents, horses, donkeys, and mules in the bodies (Abs), while another 20% exhibit reactogenicity Americas (1). VEE causes a spectrum of human diseases (5). A formaldehyde-inactivated vaccine, C-84, is well tol ranging from inapparent infection to acute encephalitis erated, but requires multiple immunization and periodic (2). Neurological disease appears in four to 14% of cases booster, and fails to provide protection against aerosol (3). The incidence of human infection during equine epi challenge in some rodent models (6). zootics could be up to 30% (4). VEE is a potential biologi VEE complexes comprise a group of antigenically cal warfare agent of concern, due to its high infectivity related, but distinct viruses divided into six subtypes (7) rate or aerosol transmissibility. However, there are no . VEE virions are composed of an icosahedral nucleocap antiviral drugs available that are effective against VEE. sid, which is surrounded by a lipid envelope containing Although live-attenuated and inactivated vaccines two structural glycoproteins, El and E2 (8) . Epitopes on El and E2 are the targets of neutralizing Abs . Studies have shown that the viral neutralizing epitopes are *T o whom correspondence should be addressed. Tel: +1-403-544 mainly located on the E2 protein, and that the E2c 4628, Fax: +1-403-544-3388, E-mail: [email protected] epitope appears to be the hub of the neutralization Vol. 133, No. 1, 2003 59 © 2003 The Japanese Biochemical Society . 60 W.-G. Hu et al. epitopes (9). Monoclonal antibody (MAb) 1A1A4 is spe cific for Etc. This MAb has been shown to be efficient in protecting animals from a lethal peripheral challenge with virulent VEE (9). Murine MAbs, however, have serious disadvantages as therapeutic agents in humans (10, 11). They induce human anti-mouse Ab responses. Re-treatment may result in rapid clearance of the murine MAbs and ana phylaxis. Limitations to the use of murine MAbs for clin ical applications led to the development of single-chain fragment variable (scFv) Abs (12). ScFv Abs have several advantages compared with the mouse parental MAbs from which they are generated. They generally retain the same specificity and similar affinity to antigens (13), and exhibit decreased immunogenicity as compared with the parental mouse MAbs (14). Furthermore, scFv Abs can be produced economically and in a short time in bacteria or yeast (15, 16), and can be manipulated by genetic engi neering to form novel proteins by fusion with other mole cules, such as metal-binding proteins (17), cytokines (18), toxins (19), and T cell receptors (20). Our laboratory has cloned and characterized several scFv Abs against genus alphavirus, VEE or Western equine encephalitis virus (WEE) (21-23). An anti-VEE Fig. 1. pRSmA116huFc construct. The mA116 scFv Ab gene A116 scFv Ab was cloned from 1A4A1 MAb (24). How was cloned into the SfiI and NotI sites of pRSlOB5huFc to replace ever, in vitro binding assays demonstrated that A116 the 10B5 scFv Ab gene as described under "MATERIALS AND scFv Ab had low binding affinity to VEE, as compared to METHODS." the parental MAb. Sequence analysis of A116 revealed that three bases were missing in conserved framework-1 of the light chain variable region (VL). PCR-based site mutagenesis (Alvi et al., submitted). The mA116 scFv Ab directed mutagenesis was used to reintroduce the three DNA fragment was separated from vector pCANTAB 5E by missing bases, resulting in a repaired A116 scFv Ab, des restriction enzyme digestion with Sfi I/Not I, followed by ignated mA116 scFv Ab (Alvi et al., submitted). This gel electrophoresis and purification with a gel extraction repaired scFv showed affinity to VEE comparable to that kit (Qiagen, Mississauga, ON). The fragment was sub of the parental 1A4A1 MAb. cloned into the SfiIlNotI site of the pRS10B5huFc expres Antiviral immunity is complex, with several factors sion vector (22), replacing the anti-WEE 10B5 scFv Ab being involved, such as the Ab response, cell-mediated gene. The fragment was inserted in-frame with the se immunity, and induction of interferon. The Ab response quence encoding 36 kDa human IgG1 CH, just before the to viruses includes not only neutralization of infectivity CHI domain. A start codon, followed by a fragment con for susceptible host cells, but also host effector functions taining a 6 His tag and X-press epitope, was added at the 5•Œ such as complement-mediated lysis of infected host cells end for ease of purification and detection. The construct, and opsonization. The host effector functions of Abs are pRSmA116huFc, was shown in Fig. 1. The recombinant attributable to the crystallizable fragment (Fc) regions plasmid DNA was transferred into E. coli BL-21 (DE3) (10). ScFv Abs lack a Fc region, and thus lack effector pLys S competent cells containing a genomic source of T7 functions. RNA polymerase under lacUV5 promoter control (Invitro In this study, we have genetically joined a truncated gen, Carlsbad, CA). The recombinant plasmid, containing human IgGI heavy chain constant region (CH) to anti the mAll6 scFv Ab gene insert, was confirmed by restric VEE mA116 scFv Ab, resulting in clone mA116huFc Ab. tion digestion and DNA sequencing. Six primers for DNA The goal of this work was to confer some of the human sequencing were made with an Oligo 1,000 DNA synthe associated effector functions on the scFv Ab without in sizer (Beckman Instruments, Fullerton, CA), and were as creasing the immunogenicity in humans. The mA116huFc follows: SC-1 (5•Œ-CATCATCATCATCATCAT-3•Œ), SC-9 (5•Œ Ab gene was expressed in E. coli, and the resulting prod CAGCAGCCAACTCAGCTT-3•Œ), SC-13 (5•Œ-ACTCCTGACA uct purified refolded in vitro, and characterized. The TCCTGTCG-3•Œ), HL-2 (5•Œ-TCTAACGGGACCAGAGAC results revealed that mA116huFc Ab retained antigen AAC-3•Œ), HL-3 (5•Œ-GGTAAGTTCAGGGACAGG-3•Œ), and binding affinity to VEE and possessed some human Ig GI HU-4 (5•Œ-TGCAAGGCCAGTCAGGATGTG-3•Œ) . The se Fc domain functions, such as recognition by protein G quencing reactions were performed with a Big Dye Termi and human Clq binding. natorTM cycle sequencing kit (PE Biosystems , Mississauga, ON) . The reaction products were purified on Centri-SepTM columns (Princeton Separations, Adelphia MATERIALS AND MATHODS , NJ), and then applied to an ABI 310 genetic analyzer system (Applied Bi Construction of pRSmA116huFc-The mA116 scFv Ab osystem, Fullerton, CA). Sequences were assembled and analyzed using Lasegene DNA software (DNA Star gene was originally derived from the A116 scFv Ab gene, , Madi which was subsequently replaced by site-directed son, WI). J. Biochem. Chimeric Antibody against VEE 61 Expression, Purification, and Refolding of the Recom mercaptoethanol) or non-reducing conditions. The bands binant mA116huFc Ab-The recombinant protein ex were visualized by Coomassie Blue staining.
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